Retrovirus capsids are unusual in assembling inside the maturing virion, not in the cytoplasm or the nucleus of the infected cell. Capsid protein is incorporated into the provirion as part of a spherical shell of the Gag polyprotein. After the provirion has budded off, the viral protease is activated and dissects Gag into its matrix (MA), capsid (CA), and nucleocapsid (NC) components. Of these, CA reassembles to form the virus capsid, housing the RNA, NC, and the replicase. Evidence suggests that a correctly formed core is essential for infectivity. In FY 08 we published a study in which cryo-electron tomography was used to visualize mature virions of Rous Sarcoma Virus (RSV), a prototypic alpha-retrovirus. Their cores were found to be highly polymorphic. We also found that RSV virions, like HIV virions, contain unassembled CA subunits; moreover, the fraction of CA that is assembled correlates with core morphology. These observations implied that initiation of capsid assembly is a critical determinant of core morphology. They also indicated that capsid polymorphism is tolerated, provided that a closed shell is produced and the viral genome and associated enzymes are correctly packaged. During FY 12, we focused on three sub-projects. One relates to aspects of capsid assembly and maturation and their implications for infectivity. The second addresses the structure and assembly properties of Rev, the HIV transactivator. The third relates to the membrane-fusing properties of envelope glycoprotein and their involvement in infection. 1) Protease inhibitors were the first drugs to be used successfully against HIV. More recently, another class of antiviral drugs has been identified that inhibits maturation differently. One of them, Beviramat (BVM), also known as PA-457, is an effective and specific drug currently in clinical trials. We investigated its mode of action by using cryo-electron tomography to determine the three-dimensional structure of virions isolated from HIV-infected cells after BVM treatment. We found that BVM-treated virions contain an incomplete shell of protein underlying the viral envelope, with a honeycomb structure similar to the Gag lattice of immature HIV but lacking the innermost layer that is associated with NC protein. These and other related findings were published in the past year (1). We have gone on to carry out similar analyses with a second maturation inhibitor, PF-46396, that is chemically unrelated to BVM. We find that, as with BVM, PF-46396 treatment results in particles containing a partial shell beneath the viral membrane, suggesting similar stabilization of the immature lattice. However, unlike BVM, a certain fraction of PF-46396-inhibited particles still contain a well formed conical core structure, suggesting a more subtle mature assembly defect than that induced by BVM. We also investigated the ability of the CA-SP1 protein (actually the Rous sarcoma virus homolog, CA-SP) to assemble in vitro. A paper reporting these studies has been submitted for publication and is currently in revision. 2) Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process, Rev oligomerizes in association with a structured RNA molecule, the Rev response element. Detailed information on the structure of Rev and on this interaction is essential for the design of antiviral drugs that impede Rev's function. For many years crystallographic studies were hampered by Rev's tendency to aggregate. However, we were able to construct a hybrid monoclonal antibody whose Fab forms a stable complex with Rev, and solve these co-crystals at 3.2 resolution. They revealed a Rev dimer, bound on either side by a Fab, where the ordered portion of Rev (residues 9-65) contains two co-planar alfa-helical hairpins. The C-terminal 40% is disordered. Subunits dimerize through overlapping of the hairpin prongs, crossing at an angle of about 140 degrees. Two papers reporting these studies were published during FY 11. Our continuing research has aimed at improving the resolution of our Rev structure. To do so, we complexed Rev with a single-chain version of the same Fab (scFv). This complex producing crystals in four different space groups that diffracted to a best resolution of 0.23 nm. They were all solved and all revealed essentially the same structure, implying that it is the solution structure although the crossing angle varies considerably. These experiments are being complemented with cryo-EM studies of the hollow helical tubes that Rev assembles into in vitro. These tubes also exhibited a limited polymorphism, mainly affecting tube diameter, that appears to have its origins in the same crossing-angle variations. The main utility of these studies is to characterize intermolecular interactions that Rev engages in in physiologically relevant complexes. 3) Retrovirus infection starts with the binding of envelope glycoproteins to host cell receptors. Subsequently, conformational changes in the glycoproteins trigger fusion of the viral and cellular membranes. Some retroviruses, such as Avian Sarcoma/Leukosis Virus (ASLV), employ a two-step mechanism in which receptor binding precedes low pH activation and fusion. We used cryo-electron tomography to study virion/receptor/liposome complexes that simulate the interactions of ASLV virions with cells. Binding the soluble receptor at neutral pH renders virions capable of binding liposomes tightly enough to alter their curvature. At virion-liposome interfaces, the glycoproteins are 3-fold more concentrated than elsewhere in the viral envelope, indicating specific recruitment to these sites. Sub-tomogram averaging shows that the oblate globular domain in the prehairpin intermediate (presumably the receptor-binding domain) is connected to both the target and the viral membrane by 2.5nm-long stalks and is partially disordered compared to its native conformation. Upon lowering the pH, fusion takes place. Fusion is a stochastic process that, once initiated, must be rapid as only final (post-fusion) products were observed. These fusion products showed glycoprotein spikes on their surface, with their interiors occupied patchily by dense material, but an absence of capsids, implying their disassembly. In addition, some presented a density layer underlying and resolved from the viral membrane, which may represent detachment of the matrix protein to facilitate the fusion process (1).